The invention relates to a method of screening a subject for a predisposition to an adverse drug reaction involving prolonged QT intervals. The genetic screening of patients for said predisposition focuses on genes associated with QT interval prolongation, including LQT genes, P-glycoprotein membrane pump proteins (P-gp), multidrug resistance genes and cytochrome P450-mediated drug metabolism genes.
I. LQT and Cytochrome P450 Genes and Polymorphisms
1. LQT Genes
Genes associated with long QT (LQT) syndrome (LQTS) include KVLQT1 (LQT1), HERG (LQT2), SCN5A (LQT3) and MinK (LQT5). A fifth gene locus exists on human chromosome 4 (e.g., LQT4). Recently, a sixth gene (LQT6) has been identified (Wang et al., Ann. Med. 30: 58–65 (1998)). All but LQT3 encode cardiac potassium ion (K+) channel proteins; LQT3 encodes a cardiac sodium ion (Na+) channel protein (Vincent, Annu. Rev. Med. 49: 263–74 (1998)). At least 180 mutations have been identified among these genes (Abbott et al., Cell 97: 175–87 (1999); Vincent. Annu. Rev. Med. 49: 263–74 (1998); Curran et al., Cell 80: 795–803 (1995); Berthet et al., Circulation 99: 1464–70 (1999); Dausse et al., J. Mol. Cell Cardiol. 28: 1609–15 (1996); Chen et al., J Biol. Chem. 274: 10113–8 (1999); and Sanguinetti et al., Proc. Natl. Acad. Sci. U.S.A. 93: 2208–12 (1996)). Some of these mutations cause altered ion channel function resulting in non-drug induced prolonged QT intervals and a propensity for Torsades de Pointes (TdP) (See, e.g., Berthet et al., Circulation 99: 1464–70 (1999)). Accordingly, genetic screening can be performed on subjects suspected of having long QT syndrome, as well as other patients (see, e.g., Satler et al., Hum. Genet. 102: 265–72 (1998)). Larson et al., Hum. Mutat. 13: 318–27 (1999) reported a high-throughout single strand polymorphism (SSCP) analysis for detecting point mutations associated with LQTS.
U.S. Pat. No. 5,599,673 claims two (e.g., HERG and SCN5A) of the six LQT genes. Two HERG-related genes have also been claimed (U.S. Pat. No. 5,986,081). International PCT Application WO 97/23598 describes a method of assessing a patient's risk for long QT syndrome (LQTS) by screening for genetic mutations in the MinK gene. However, these patents do not disclose methods of diagnosing a patient's predisposition to an adverse drug reaction involving elongation of the QT interval due to mutations in any of the LQT genes.
Drugs have been identified that cause QT interval prolongation, and thereby adverse drug reactions. Certain antihistamines, such as terfenadine (e.g., Seldane®) and astemizole (e.g., Hismanal®), reportedly block potassium channels (Woosley, Annu. Rev. Pharmacol. Toxicol. 36: 233–52 (1996)) and inhibit the HERG protein, and thereby were postulated to induce Torsades de Pointes (Wang et al., 1998). All antiarrhythmic drugs that lengthen repolarization reportedly can cause Torsades de Pointes (Drici et al., Circulation 94: 1471–4 (1996)). Additional non-cardiac and cardiac drugs capable of inducing QT prolongation including many that were identified by the inventor were released on Mar. 27, 1998 at the following web site: www.qtdrugs.org. However, Wei et al., Circulation 92:1–125 (1995) could not identify HERG or SCN5A gene mutations that were linked to acquired LQTS in patients treated with an anti-arrhythmic agent. To the best knowledge of the inventor, no one has described diagnosing a predisposition towards an adverse drug or drug-drug reaction which causes QT interval elongation by screening patients for one or more polymorphisms in one or more LQT genes.
1. Cytochrome P450 Genes
The cytochrome P450 enzymes have also been linked to adverse drug reactions. CYP2D6 was the first cytochrome P450 isoform found to be genetically polymorphic in its distribution (Eichelbaum et al., Eur. J. Clin. Pharmacol. 16: 183–7 (1979); and Mahgoub et al., Lancet 2: 584–6 (1977)), and it is now clear that this enzyme metabolizes a large number of drugs (Inaba et al., Can. J. Physiol. Pharmacol. 73: 331–8 (1995); and Buchert et al., Pharmacogenetics 2: 2–11 (1992)). At least 30 mutations exist which alter the activity or specificity of CYP2D6 (Jordan et al., Endocr. Rev. 20: 253–78 (1999)). These include alleles that contain single point mutations resulting in no activity (e.g., CYP2D6*4), alleles in which the CYP2D6 gene has been deleted (e.g., CYP2D6*5) and alleles in which it has been duplicated (e.g., CYP2D6*2_n) (Aklillu et al., J. Pharmacol. Exp. Ther. 278: 441–6 (1996)).
There are numerous cytochrome P450 genes which are involved in the metabolism of drugs and drug metabolites. Several of them include CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7. Allelic variations exist amongst these genes. Certain of these allelic variations combine to produce a poor metabolizer phenotype in 7% of Caucasians, but smaller percentages of Africans and Asians and the “ultrarapid” phenotype in ˜5% of Caucasian and up to 30% Africans. As ethnic-specific alleles for both Asians (Yokoi et al., Pharm. Res. 15: 517–24 (1998)) and Africans (Aklillu et al., J. Pharmacol. Exp. Ther. 278: 441–6 (1996); and Oscarson et al., Mol. Pharmacol. 52: 1034–40 (1997)) have been identified, that may alter the mean activity of the enzymes in these populations (see Table 1 below), it is also important to test for these alleles in studies of the relationship between genotype and phenotype.
TABLE 1Chromosome Distribution of Cytochrome P450 GeneChr.Chr. 10Chr. 10Chr. 2215PolymorphicPolymorphicPolymorphicChr. 10Chr. 73–5%1–3%5–10%CaucasianCaucasianCaucasianPMsPMsPMs15–20% AsianPMsIn fact, due to the metabolic differences, methods have been reported which identify a drug which interacts with the CYP2C19 gene product, S-mephenytoin 4′-hydroxylase (U.S. Pat. No. 5,786,191).
Methods for detecting the presence or absence of mutations in certain of the cytochrome P450 genes have been described. For example, U.S. Pat. No. 5,891,633 relates to a method of identifying mutations in the cytochrome P450 genes CYP2C9 and CYP2A6.
International PCT Application WO 95/30772 reportedly describes a CYP2D6 gene polymorphisms involving a 9 bp insertion in exon 9, which is associated with a slower than normal rate of drug metabolism in individuals bearing it and may be therefore useful diagnostically. PCR primers have been described for detecting mutations in drug metabolism enzymes, including detection of the debrisoquine polymorphism, mephenytoin polymorphism and the acetylation polymorphism (U.S. Pat. Nos. 5,648,484 and 5,844,108). Additional mutations have been identified in CYP2D6 bufuralol-1′-hydroxylase, including mutations at positions 271, 281, 294, and 506 which result in metabolizer/poor metabolizer phenotypes as described in International PCT Application WO 91/10745 and U.S. Pat. No. 5,981,174.
Japanese Patent No. 8168400 provides a method of determining mutations in exons 6 and 7 of the CYP2C19 gene. Japanese Patent No. 10014585 describes primers and methods of detecting a mutation in exon 5 of CYP2C19, which is related to the abnormal metabolism of diazepam, imipramine, omeprazole and propranolol. U.S. Pat. No. 5,912,120 claims a method of diagnosing a patient having a deficiency in S-mephenytoin 4′-hydroxylase activity by detecting polymorphisms at nucleotides 681 or 636.
U.S. Pat. No. 5,719,026 provides methods and primers for detecting a polymorphisms in CYP1A2 and assessing the changes in the drug activity of theophylline associated with those polymorphisms.
Japanese Patent No. 10286090 reportedly describes methods and primers to detect mutations in CYP2E1. These mutations are reported as being useful for determining the safety margin for drug administration for the treatment or related diseases.
Despite these teachings and to the best of the inventor's knowledge, no one has described or suggested that a combination of polymorphisms in LQT and cytochrome P450 genes can induce acquired LQTS in a subject in response to the administration of a drug or drugs.
C. P-glycoprotein Pump Genes
P-Glycoprotein Pump (P-gp) in the development of drug-resistant tumor cells has been extensively studied (Lo et al., J. Clin. Pharmacol. 39: 995–1005 (1999)). P-gp is an ATP-dependent drug pump that extrudes a broad range of cytotoxic agents from the cells end is encoded by a gene called MDR-1, for multidrug resistance (Loo et al., Biochem. Cell. Biol. 77: 11–23 (1999); and Robert, Eur. J. Clin. Invest. 29: 536–45 (1999)). The human P-gp sequence has been described by Chen et al., Cell 47: 381–9 (1986) and has the GenBank Accession No. M14758.
Its physiological role may be to protect the body from endogenous and exogenous cytotoxic agents. The protein has clinical importance because it contributes to the phenomenon of multidrug resistance during chemotherapy (Loo et al., 1999) and the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells (Ambudkar et al., Annu. Rev. Pharmacol. Toxicol. 39: 361–98 (1999)). Specifically, the over expression of this membrane pump appears to extrude many xenobiotics out of the tumor cells (Robert, 1999). However, considerable controversy remains about the mechanism of action of this efflux pump and its function in normal cells (Ambudkar et al., 1999).
Multidrug resistance (MDR) can be diagnosed in tumors using molecular biology techniques (e.g., gene expression at the mRNA level), by immunological techniques (e.g., quantification of the P-glycoprotein itself) or by functional approaches (e.g., measuring dye exclusion) (Robert, 1999).
Drugs have been developed which reverse or modulate MDR. For example, PSC-833 is a non-immunosuppressive cyclosporin derivative that potently and specifically inhibits P-gp (Atadja et al., Cancer Metastasis Rev. 17: 163–8 (1998)). Also, compounds have been identified which increase or modulate the bioavailability of pharmaceutical compounds. See, e.g., U.S. Pat. Nos. 6,004,927; 5,962,522; 5,916,566; 5,716,928; 5,665,386; and 5,567,592. P-gp activity has been altered by expression of antisense nucleotides specific to MDR-1 (U.S. Pat. No. 6,001,991). Methods and assays have also been developed which assess whether multidrug resistance has been reversed (U.S. Pat. No. 5,403,574).
Mutations have also been identified which alter an agents interaction with P-gp. For instance U.S. Pat. No. 5,830,697 discloses single and double mutations (Phe335 and/or 336) which alters the spectrum of cross-reactivity to cytotoxins and resistance to modulation by cyclosporins. Another mutation at V185G in P-gp confers increased resistence to colchicine (U.S. Pat. No. 5,830,697). P-gp sensitivity to vinblastine, colchicine, VP16 and adriamycin, common chemotherapeutic agents, was up- and down-regulated by altering 61His to another amino acid residue (Taguchi et al., Biochemistry 36: 8883–9 (1997)). Moreover, different drugs interact differently with P-gp and mutated forms of P-gp, such that one mutation may influence the activity of one drug and not another (See, e.g., Chen et al., J. Biol. Chem. 272: 5974–82 (1997); Bakos et al., Biochem. J. 323: 777–83 (1997); and Gros et al., Proc. Natl. Acad. Sci. USA 88: 7289–93 (1991)). However, despite the information regarding the influence such mutations may have on drug activity, no association has been made linking P-gp by itself or in combination with another protein in influencing QT intervals or inducing Torsades de Pointes.
II. Nucleic Acid Hybridization
The capacity of a nucleic acid “probe” molecule to hybridize (i.e., base pair) to a complementary nucleic acid “target” molecule forms the cornerstone for a wide array of diagnostic and therapeutic procedures Hybridization assays are extensively used in molecular biology and medicine. Methods of performing such hybridization reactions are disclosed by, for example, SAMBROOOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), HAYMES ET AL., NUCLEIC ACID HYBRIDIZATION: A PRACTICAL APPROACH (IRL Press, Washington, D.C. (1985)) and KELLER ET AL., DNA PROBES (2nd Ed., Stockton Press, New York (1993)).
Many hybridization assays require the immobilization of one component to a solid support. Nagata et al., FEBS Letters 183: 379–82 (1985) described a method for quantifying DNA which involved binding unknown amounts of cloned DNA to microtiter wells in the presence of 0.1 M MgCl2. A complementary biotinylated probe was then hybridized to the DNA in each well and the bound probe measured colorimetrically. Dahlen et al., Mol. Cell. Probes 1: 159–168 (1987) have discussed sandwich hybridization in microtiter wells using cloned capture DNA adsorbed to the wells. An assay for detecting HIV-1 DNA using PCR amplification and capture hybridization in microliter wells also has been reported (Keller et al., J. Clin. Microbial. 29: 638–41 (1991)). The NaCl-mediated binding of oligomers to polystyrene wells has been discussed by Cros et al. (French Patent No. 2,663,040) and by Nikiforov et al., PCR Methods Applic. 3: 285–291 (1994). A cationic detergent-mediated binding of oligomers to polystyrene wells has been described by Nikiforov et al., Nucleic Acids Res. 22: 4167–75 (1994).
III. Analysis of Single Nucleotide DNA Polymorphisms
Many genetic diseases and traits (i.e. hemophilia, sickle-cell anemia, cystic fibrosis, etc.) reflect the consequences of mutations that have arisen in the genomes of some members of a species through mutation or evolution (Gusella, Ann. Rev. Biochem. 55: 831–54 (1986)). In some cases, such polymorphisms are linked to a genetic locus responsible for the disease or trait; in other cases, the polymorphisms are the determinative characteristic of the condition.
Single nucleotide polymorphisms (SNPs) differ significantly from the variable nucleotide type polymorphisms (VNTRs), that arise from spontaneous tandem duplications of di- or tri-nucleotide repeated motifs of nucleotides (Weber, U.S. Pat. No. 5,075,217; Armour et al., FEBS Lett. 307: 113–5 (1992); Horn et al., PCT Application No. WO 91/14003; Moore et al., Genomics 10: 654–60 (1991); Hillel et al., Genet. 124: 783–9 (1990)), and from the restriction fragment length polymorphisms (“RFLPs”) that comprise variations which alter the lengths of the fragments that are generated by restriction endonuclease cleavage (e.g., Fischer et al., (PCT Application No. WO 90/13668); and Uhlen (PCT Application No. WO 90/11369)).
Because SNPs constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation; it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses a method for determining the identity of the nucleotide present at a particular polymorphic site that employs a specialized exonuclease-resistant nucleotide derivative.
Cohen et al. (French Patent 2,650,840; and PCT Application No. WO 91/02087) discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3′ to a polymorphic site.
Additional SNP detection methods include the Genetic Bit Analysis method described by Goelet et al. (PCT Application No. 92/15712). The method of Goelet et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3′ to a polymorphic site.
Cheesman (U.S. Pat. No. 5,302,509) describes a method for sequencing a single stranded DNA molecule using fluorescently labeled 3′-blocked nucleotide triphosphates. An apparatus for the separation, concentration and detection of a DNA molecule in a liquid sample has been described by Ritterband et al. (PCT Patent Application No. WO 95/17676).
Several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Prezant et al., Hum. Mutat. 1: 159–64 (1992); Ugozzoli et al., GATA 9: 107–12 (1992); and Nyren et al., Anal. Biochem. 208: 171–5 (1993)).
IV. Methods of Immobilization Nucleic Acids to a Solid-Phase
Several of the above-described methods involve procedures in which one or more of the nucleic acid reactants are immobilized to a solid support. Currently, 96-well polystyrene plates are widely used in solid-phase immunoassays. PCR product detection methods that use plates as a solid support and DNA chips have been described. The microtiter plate method requires the immobilization of a suitable oligonucleotide probe into the microtiter wells, followed by the capture of the PCR product by hybridization and colorimetric detection of a suitable hapten.
Covalent disulfide bonds have been previously used to immobilize both proteins and oligonucleotides. Chu et al. (Nucl. Acids Res. 16: 3671–91 (1988)) discloses a method for coupling oligonucleotides to nucleic acids or proteins via cleavable disulfide bonds.
Gentalen et al., Nucl. Acids Res. 27: 1485–91 (1999) describe a cooperative hybridization method lo establish physical linkage between two loci on a DNA strand by using hybridization to a new type of high-density oligonucleotide array. This same method can be used to determine SNP haplotypes.
Yershov et al., Proc. Natl. Acad. Sci. USA 93: 4913–8 (1996) describe an oligonucleotide microchip which has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. This technology was suggested for large scale diagnostics in gene polymorphism studies.
Guo et al., Nucl Acids Res. 22: 5456–65 (1994) describe a simple method for the analysis of genetic polymorphisms allele-specific oligonucleotide raised bound to glass supports. This method was demonstrated in parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.
More recently. Gilles et al., Nat. Biotechnol . 17: 365–70 (1999) have described a rapid assay for SNP detection utilizing electronics circuitry on silicon Microchips. Holloway et al., Hum. Mutat. 14: 340–7 (1999) also compares methods for high-throughput SNP typing using TaqMan® liquid phase hybridization, PCR-SSOP or ARMS-microplate array diagonal gel electrophoresis (MADGE).
As the world population ages and new drugs are identified, more and more patients will administer one or more pharmaceutical compositions, such that an individual drug or drugs combination can cause adverse drug reactions. Therefore, not withstanding what has been previously reported in the literature, the inventor herein describes methods and compositions for diagnosing drug interactions which involve at least one mutation in a LQT gene. Additional mutations may exist in certain cytochrome 450 genes and P-glycoprotein pumps, which work in concert with a LQT gene mutation or other ion channel (e.g., K+or Na+) gene polymorphisms to produce an adverse drug or drug-drug reaction. The specification also discloses kits and compositions for diagnosing a subject's predisposition to QT interval elongation in response to the administration of one or more pharmaceutical agents.